Synthesis and in vitro antimicrobial screening of new pyrano[4,3-b]pyrane derivatives of 1H-pyrazole

Article abstract of DOI:10.1016/j.cclet.2011.09.012

A new series of pyrano[4,3-b]pyrane 4a-l bearing 1H-pyrazole has been synthesized by one pot base catalyzed cyclocondensation reaction of 1H-pyrazole-4-carbaldehyde 1a-l, malononitrile 2 and 4-hydroxy-6-methylpyrone 3. All the synthesized compounds were screened against six bacterial pathogens, namely B. subtilis, C. tetani, S. pneumoniae, S. typhi, V. cholerae, E. coli and antifungal activity against, two fungal pathogens, A. fumigatus and C. albicans using broth microdilution MIC method. Some of the compounds are found to be equipotent or more potent than that of commercial drugs, against most of employed strains.

Full text of DOI:10.1016/j.cclet.2011.09.012

Available online at www.sciencedirect.com
Chinese Chemical Letters 23 (2012) 57–60
www.elsevier.com/locate/cclet
Synthesis and in vitro antimicrobial screening of new
pyrano[4,3-b]pyrane derivatives of 1H-pyrazole
*
Chetan B. Sangani , Divyesh C. Mungra, Manish P. Patel, Ranjan G. Patel
Department of Chemistry, Sardar Patel University, Vallabh Vidyanagar 388120, Gujarat, India
Received 22 June 2011
Available online 8 November 2011
Abstract
A new series of pyrano[4,3-b]pyrane 4a–l bearing 1H-pyrazole has been synthesized by one pot base catalyzed cyclocondensa-
tion reaction of 1H-pyrazole-4-carbaldehyde 1a–l, malononitrile 2 and 4-hydroxy-6-methylpyrone 3. All the synthesized
compounds were screened against six bacterial pathogens, namely B. subtilis, C. tetani, S. pneumoniae, S. typhi, V. cholerae,
E. coli and antifungal activity against, two fungal pathogens, A. fumigatus and C. albicans using broth microdilution MIC method.
Some of the compounds are found to be equipotent or more potent than that of commercial drugs, against most of employed strains.
# 2011 Chetan B. Sangani. Published by Elsevier B.V. on behalf of Chinese Chemical Society. All rights reserved.
Keywords: Pyranopyrane; MCR; Pyrazole-4-carbaldehyde; Antimicrobial activity
An alarming increment in pathogenic resistance to existing first line standard drugs is a serious problem in
antimicrobial cure and necessitates continuing research into new classes of antimicrobials [1]. Moreover, the
progression of drug-resistant strains has contributed to the inefficiency of the straight antimicrobial therapy. This crop
up an enormous interest in antibacterial research and we strongly believe that there is an urgent call for development of
new drugs with divergent and unique structure and with a probably unusual mechanism of action from that of existing
first line drugs. Consequently, this spot of research is accorded an immense significance and keeps on attracting much
attention of increasing number of medicinal chemists.
Recently, we havebeenparticularlyinterestedinthesynthesisofN-arylpyrazoleand pyraneincorporatingstructuresfor
antimicrobial evaluations [2] on the premise that N-arylpyrazole is chemically useful synthons bearing diverse biological
activities like antimicrobial [3–5], anti-inflammatory (COX-2 inhibitor and ulcerogenic activity) [4], antitubercular [5],
antitumor [6,7], antiangiogenesis [7], anti-parasitic [8], antiviral [9], analgesic and anxiolytic activity [10]. Moreover, the
pyran nucleus is a fertile source of biologically important molecules possessing a wide spectrum of biological and
pharmacological activities, such as antimicrobial [11], antiviral [12], antiproliferative [13], antitumor [14], etc.
Multi component reaction (MCR) approach emerged as the most suitable protocol for the synthesis of
functionalized organic compounds due to the fact that the synthesis can be performed without the isolation of the
intermediates, without discharging any functional groups within short reaction time [15]. This synthetic strategy is
fruitfully employed to achieve new pyrano[4,3-b]pyran derivatives of aryloxypyrazole.
* Corresponding author.
E-mail address: chetansangani1986@yahoo.com (C.B. Sangani).
1001-8417/$ – see front matter # 2011 Chetan B. Sangani. Published by Elsevier B.V. on behalf of Chinese Chemical Society. All rights reserved.
doi:10.1016/j.cclet.2011.09.012

58
C.B. Sangani et al. / Chinese Chemical Letters 23 (2012) 57–60
Thus, in view of biological significance of pyrane, a modification on the 4-position on pyrano[4,3-b]pyrane by 3-
methyl-5-phenoxy-1-phenyl-1H-pyrazole is undertaken to check whether it may bring significant changes in
bioactivities of pyrane derivatives. As a part of our current studies in developing new antimicrobial agents via
combination of two therapeutically active moieties [16], we have synthesized and report herein pyrano[4,3-b]pyrane
derivatives 4a–l via MCR approach.
1. Experimental
All the reagents were obtained commercially and used with further purification. All melting points were taken in
open capillaries in a paraffin bath and are uncorrected. The monitoring of the progress of all reactions and
homogeneity of the synthesized compounds was carried out by thin layer chromatography (TLC). TLC was run using
TLC aluminum sheets silica gel 60 F254 (Merck). Elemental analysis (%C, H, N) was carried out by Perkin-Elmer
2400 series-II elemental analyzer (Perkin-Elmer, USA). The IR spectra were recorded in KBr on a Perkin-Elmer
1
Spectrum GX FT-IR spectrophotometer (Perkin-Elmer, USA). H NMR and 13C NMR spectra were recorded in
DMSO-d6 on a Bruker Avance 400F (MHz) spectrometer (Bruker Scientific Corporation Ltd., Switzerland) using
solvent peak as internal standard at 400 MHz and 100 MHz, respectively. Mass spectra were scanned on a Shimadzu
LCMS 2010 spectrometer (Shimadzu, Tokyo, Japan) (Scheme 1).
1.1. General procedure
A mixture of an appropriate 1H-pyrazole-4-carbaldehyde 1a–l (30 mmol), malononitrile 2 (30 mmol) and 4-
hydroxy-6-methylpyrone 3 (30 mmol) in ethanol (20 mL) containing catalytic amount of piperidine was stirred under
reflux for 1–1.5 h. On completion of reaction, monitored by TLC (ethyl acetate:hexane = 1:1), the reaction mixture
was cooled to room temperature, the solid separated was filtered and washed well with ethanol to obtain the pure
compounds 4a–l. Physical, analytical and spectroscopic characterization data of the synthesized compounds 4a and 4k
are given below:
2-Amino-4-[3-methyl-5-phenoxy-1-phenyl-1H-pyrazol-4-yl]-7-methyl-5-oxo-4,5-dihydropyrano[4,3-b]pyran-3-
carbonitrile (4a): Yield: 80%; mp: 230–231 8C; IR (KBr, n, cmÀ1): 3400 & 3180 (asym. & sym. str. of –NH2), 2205
(–CBBN str.), 1710 (–C O str.); 1H NMR (400 MHz, DMSO-d6): d 2.00, 2.33 (s, 3H, 2 Â CH3), 4.28 (s, 1H, H4),
5.68 (s, 1H, H8), 6.57–7.54 (m, 12H, NH2 + Ar–H). 13C NMR (100 MHz, DMSO-d6): d 13.07, 19.52 (CH3), 25.88
(C4), 56.24 (C–CN), 97.95 (4a), 99.17 (C8), 110.82, 114.56, 120.03, 121.63, 123.38, 127.05, 129.63, 130.17,
138.09, 145.35, 148.07, 156.20, 158.36, 158.63, 161.82 (Ar–C), 162.55 (C O); Anal. Calcd. for C26H20N4O4
(452.46): C, 69.02; H, 4.46; N, 12.38%. Found: C, 69.17; H, 4.35; N, 12.22%; MS m/z: 453.52 [M+1]+.
H3C
CHO
O
H3C
CHO
Cl
H3C
OH
R2
N
N
O
N
DMF
POCl
N
N
DMF
K CO
N
3
2
3
R2
R1
4a: R1 = H, R2 = H
4b: R1 = H, R2 = CH3
4c: R1 = H, R2 = OCH3
4d: R1 = H, R2 = Cl
R1
R1
R1
1a-l
4e: R1 = CH3, R2 = H
4f: R1 = CH3, R2 = CH3
4g: R1 = CH3, R2 = OCH3
4h: R1 = CH3, R2 = Cl
4i: R1 = Cl, R2 = H
4j: R1 = Cl, R2 = CH3
4k: R1 = Cl, R2 = OCH3
4l: R1 = Cl, R2 = Cl
CHO
O
H3C
N
N
O
R2
H3C
N
NC
NC
2
N
O
O
Ethanol
Piperidine
O
+
CN
O
H3C
OH
R2
H3C
O
4a-l
NH2
R1
3
1a-l
Scheme 1. Synthetic pathway for the compounds 1a–l and 4a–l.

C.B. Sangani et al. / Chinese Chemical Letters 23 (2012) 57–60
59
2-Amino-4-[1-(3-chlorophenyl)-5-(4-methoxyphenoxy)-3-methyl-1H-pyrazol-4-yl]-7-methyl-5-oxo-4,5-dihydro-
pyrano[4,3-b]pyran-3-carbonitrile (4k): Yield: 84%; mp: 240–241 8C; IR (KBr, n, cmÀ1): 3410 & 3195 (asym. &
1
sym. str. of –NH2), 2195 (–CBB N str.), 1705 (–C O str.); H NMR (400 MHz, DMSO-d6): d 2.03, 2.32 (s, 3H,
2 Â CH3), 3.66 (s, 3H, OCH3), 4.28 (s, 1H, H4), 5.75 (s, 1H, H8), 6.53–7.61 (m, 10H, NH2 + Ar–H). 13C NMR
(100 MHz, DMSO-d6): d 13.10, 19.41 (CH3), 25.83 (C4), 55.82 (OCH3), 56.01 (C–CN), 98.02 (4a), 99.04 (C8),
111.22, 115.20, 115.42, 119.57, 119.97, 120.92, 126.68, 131.42, 133.98, 139.22, 146.11, 148.86, 149.76, 155.52,
158.38, 158.63, 161.80 (Ar–C), 162.58 (C O); Anal. Calcd. for C27H21ClN4O5 (516.93): C, 62.73; H, 4.09; N,
10.84%. Found: C, 62.95; H, 4.26; N, 10.99%; MS m/z: 517.88 [M+1]+.
2. Results and discussion
The targeted title compounds pyrano[4,3-b]pyranes 4a–l were prepared in moderate to good yield (75–90%) by the
reaction of 3-methyl-5-aryloxy-1-aryl-1H-pyrazole-4-carbaldehydes, malononitrile and 4-hydroxy-6-methylpyrone
in refluxing ethanol containing piperidine as a basic catalyst. The required 1-aryl-5-chloro-3-methyl-1H-pyrazole-4-
carbaldehydes were prepared according to literature procedure [18] and 3-methyl-5-aryloxy-1-aryl-1H-pyrazole-4-
carbaldehydes 1a–l by our literature procedure [2a]. The reaction was carried out in aqueous media and under neutral
conditions but failed to proceed even on prolong refluxing. The reaction was also attempted under microwave
irradiation but not succeeded. The reaction proceeded in acetonitrile, methanol, benzene or DMF, in the presence of
morpholine or K2CO3 but required prolong refluxing and resulted only in poor yield. Hence, these conditions were
considered as the most optimized condition for the synthesis of title derivatives.
The plausible mechanism for the formation of the pyrane derivatives 4a–l is outlined in Scheme 2. The reaction
may proceed via an in situ initial formation of the hetarylidene nitrile, containing the electron-poor C C double bond,
from the Knoevenagel condensation between 3-methyl-5-aryloxy-1-arylpyrazole-4-carbaldehyde and malononitrile
by loss of water molecules. Finally, Michael addition of 3 to the initially formed unsaturated nitrile, i.e. nucleophilic
attack of hydroxyl moiety to the cyano olefins afforded cyclized pyrano[4,3-b]pyrane derivatives 4a–l.
1H NMR (DMSO-d6) spectrum of 4a exhibited a singlet peak around d 4.28, 5.68 stands for H4 and H8 respectively.
Amine and aromatic protons of 4a resonate as multiplets at around d 6.57–7.54. Singlets around d 2.00, 2.33 stands for
methyl protons. 13C NMR of 4a exhibited distinctive signals around d 13.07, 19.52 stands for methyl carbons and d
25.88, 56.24 stands for C4 and C–CN respectively. Aromatic carbons of 4a showed signals around d 110.82–161.82 in
the 13C NMR spectra. Moreover, distinctive signals d 162.55 stands for carbonyl carbon. The IR spectrum of
compound 4a exhibited characteristic absorption bands around 3400–3180 cmÀ1 and 2205 cmÀ1 stands for (asym. &
sym. str.) –NH2 and –CN functional groups respectively. The characteristic absorption band of lactone carbon is
observed around 1710 cmÀ1
.
The elemental analysis values and mass spectral data are in good agreement with that of theoretical data. Similarly,
all these compounds were characterized on the basis of spectral studies. All the compounds 4a–l were screened for
their antimicrobial activities using ampicillin, nystatin and griseofulvin as standard drugs by broth microdilution
method as recommended by NCCLS [17]. Reviewing the antimicrobial activity data (Table 1), majority of compounds
are found to be active against Gram-positive bacteria B. subtilis, C. tetani and a fungal pathogen C. albicans. It is worth
mentioning that minor change in the molecular configuration of these compounds profoundly influences the activity.
Ar
Pyz
Pyz
CN
CN
2
Pyz
CHO
NC
N
HO
3
NC
Ar
-H2O
Pyz
C
CN
O
H
1a-l
Hetarylidene nitrile
Pyz
Pyz
H
H
NC
H2N
3
2
5
H
NC
HN
NC
N
4
Ar
Ar
Ar
C
C
O
6
O
O
4a-l
Scheme 2. Plausible mechanistic pathway for the compounds 4a–l.

60
C.B. Sangani et al. / Chinese Chemical Letters 23 (2012) 57–60
Table 1
Antimicrobial activity of compounds 4a–l.
Compound
Minimum inhibitory concentration, MIC (mg/mL)
Gram-positive bacteria
Gram-negative bacteria
Fungal species
B. subtilis
C. tetani
S. pneumoniae
E. coli
S. typhi
V. cholerae
A. fumigatus
C. albicans
4a
4b
4c
4d
4e
4f
4g
4h
4i
200
200
250
500
500
100
200
500
500
250
250
200
250
–
200
200
500
500
250
250
250
200
250
250
250
500
250
–
250
200
250
100
500
500
200
100
250
250
500
200
100
–
100
62.5
500
250
200
250
200
500
500
200
62.5
500
100
–
100
250
500
250
250
500
250
500
500
250
100
200
100
–
250
100
500
250
200
250
200
250
500
200
100
500
100
–
1000
500
500
250
250
1000
250
500
500
100
250
200
500
500
–
500
200
500
1000
>1000
>1000
500
4j
4k
4l
500
1000
>1000
–
A
B
100
100
500
C
–
–
–
–
–
–
100
A, ampicillin; B, nystatin; C, griseofulvin; ‘–’, ‘not tested’.
Acknowledgment
The authors are grateful to the Department of Chemistry, Sardar Patel University, India for providing laboratory
facilities.
References
[1] N. Woodford, Expert Opin. Investig. Drugs 12 (2003) 117.
[2] (a) C.B. Sangani, D.C. Mungra, M.P. Patel, et al. Central Eur. J. Chem. 9 (2011) 635;
(b) N.J. Thumar, M.P. Patel, ARKIVOC xii (2009) 363.
[3] I. Damljanovic, M. Vukicevic, N. Radulovic, et al. Bioorg. Med. Chem. Lett. 19 (2009) 1093.
[4] A.A. Bekhit, H.M.A. Ashour, Y.S.A. Ghany, et al. Eur. J. Med. Chem. 43 (2008) 456.
[5] P.T. Chovatia, J.D. Akabari, P.K. Kachhadia, et al. J. Serb. Chem. Soc. 71 (2007) 713.
[6] M.D. Joksovic, V. Markovic, Z.D. Juranic, et al. J. Organomet. Chem. 694 (2009) 3935.
[7] A.H. Abadi, A.A.H. Eissa, G.S. Hassan, Chem. Pharm. Bull. 51 (2003) 838.
[8] P. Rathelot, N. Azas, H. El-Kashef, et al. Eur. J. Med. Chem. 37 (2002) 671.
[9] A.I. Hashem, A.S.A. Youssef, K.A. Kandeel, et al. Eur. J. Med. Chem. 42 (2007) 934.
[10] S.C. Shetty, V.C. Bhagat, Asian J. Chem. 20 (2008) 5037.
[11] M.M. Khafagy, A.H.F.A. El-Wahas, F.A. Eid, et al. Farmaco 57 (2002) 715.
[12] A.G. Martinez, L.J. Marco, Bioorg. Med. Chem. Lett. 7 (1997) 3165.
[13] C.P. Dell, C.W. Smith, Eur. Pat. Appl. 537,949 (1993);
C.P. Dell, C.W. Smith, Chem. Abstr. 119 (1993) 139102d.
[14] S.J. Mohr, M.A. Chirigos, F.S. Fuhrman, et al. Cancer Res. 35 (1975) 3750.
[15] L. Boulard, S. BouzBouz, J. Cossy, et al. Tetrahedron Lett. 45 (2004) 6603.
[16] (a) D.C. Mungra, M.P. Patel, D.P. Rajani, et al., Eur. J. Med. Chem., in press;
(b) C.B. Sangani, D.C. Mungra, M.P. Patel, et al. Central Eur. J. Chem. 9 (2011) 635;
(c) J.A. Makawana, M.P. Patel, R.G. Patel, Med. Chem. Res., in press;;
(d) N.M. Shah, M.P. Patel, R.G. Patel, J. Heterocycl. Chem., in press;;
(e) J.A. Makawana, M.P. Patel, R.G. Patel, Chem. Papers, in press;
(f) D.C. Mungra, M.P. Patel, R.G. Patel, Med. Chem. Res. 20 (2011) 782;
(g) D.C. Mungra, M.P. Patel, R.G. Patel, ARKIVOC xiv (2009) 64;
(h) N.K. Ladani, M.P. Patel, R.G. Patel, ARKIVOC x (2009) 292.
[17] National Committee for Clinical Laboratory Standards (NCCLS), 940, West Valley Road, Suite 1400, Wayne, PA 19087-1898, USA, Performance
Standards for Antimicrobial Susceptibility Testing, Twelfth Informational Supplement (ISBN: 1-56238-454-6), M100-S12 (M7) (2002).
[18] R.A. Pawar, A.A. Patil, Indian J. Chem. 33B (1994) 156.